The production of effective and safe vaccines against harmful viruses and other pathogens continues to be a difficult endeavor. To date, the most successful vaccines involve the use of inactivated viruses or live attenuated viruses obtained from multiple passages of wild-type viruses in tissue culture or in non-human primates. A concern that still lingers, however, is that outbreaks occasionally occur, apparently from improper inactivation of the viruses, reversion or pseudoreversion of the viruses to virulent strains, extension of the host range, and/or contamination of vaccines with live virus.
To overcome some of these complications, considerable research effort has been expended to examine the feasibility and efficacy of immunizing with empty viral capsids and pathogen-derived proteins and peptides. Unfortunately, the antigenicity profiles for complex virions and empty capsids are often quite different. This phenomenon has been documented for several picornaviruses, including rhinovirus (Lonberg-Holm & Yin, J. Virol., 12:114-123, 1973), poliovirus (Mayer, et al., J. Immunol., 78:435-455, 1957), foot-and-mouth disease virus (FMDV) (Rowlands, et al., J. Gen. Virol., 26:227-238, 1975), and Coxsackie B virus (Frommhagen, J. Immunol., 95:818-822, 1965). Studies with individual virion proteins have shortcomings as well. Individual coat proteins, for instance, have antigenic determinants absent from intact viruses (Wiegers & Derrick, J. Gen. Virol., 64:777-785, 1983) and are generally far less effective at stimulating neutralizing antibodies than are whole virions. Attempts to use peptides to provide protection against dangerous pathogens have also been disappointing. Despite occasional examples of success (e.g., Bittle, et al., Nature, 298:30-33, 1982; Pfaff, et al., EMBO. J., 1:869-874, 1982), most peptides fail to protect vaccinated animals, even when they are capable of stimulating the production of neutralizing antibodies (e.g., Ada & Skehel, Nature, 316:764-765, 1985; Tiollais, et al., Nature, 317:489-495, 1985; DiMarchi, et al., Science, 232:639-641, 1986).
A more recent approach to vaccine development and the one which is utilized in the present invention uses chimeric viruses or virus-like particles (VLPs) as vehicles for presentation of foreign antigens to the immune system. A number of virus-like particles (VLPs) composed of fusion proteins have been shown to test positively in standard enzyme-linked immunosorbent assays (ELISAs) with antibodies directed against either substituent of the fusion protein. Among chimeric VLPs, almost none have been tested for their ability to protect infected animals; an exceptional case involved the testing of a hepatitis B surface antigen:poliovirus VPI chimera which, when injected into mice, produced only weak protection against poliovirus (Delpeyroux, et al., Science, 233:472-475, 1986). Live recombinant viruses with the composite antigenicity of mixed poliovirus types have recently been produced in other laboratories (Kohara, et al., J. Virol., 62:2828-2835, 1988; Martin, et al., EMBO. J., 7:2839-2847, 1988; Burke, et al., J. Gen. Virol., 70:2475-2479, 1989); these reports gave no reference to testing of these live chimeras for protection against the virus. In another experiment, Evans, et al., (Nature, 339:385-388, (1989)) incorporated an epitope (positions 735-752) from the transmembrane glycoprotein of HIV-1, gp41, into the neutralizing antigenic region of VPI (NAg-1) of poliovirus 1 Sabin. These workers reported that rabbit antisera and monoclonal antibodies elicited by the chimera were capable of neutralizing in vitro a wide range of American and African isolates of HIV-1. Protection studies were not reported with this construct.
Unfortunately, previously developed chimeric viruses, such as those based on poliovirus and vaccinia virus, have certain characteristics which make them less than ideal. One of the most significant drawbacks to these chimeric viruses is that their effectiveness as vaccines is limited since many individuals already have a significant immune response to the native virus. Also, at least in the case of poliovirus, the use of a live vaccine carries with it the fact that the native virus is a major pathogen with associated risks.
A cDNA clone of HRV14 was reported by Mizutani and Colonno (J. Virol., 56:628, 1985). This clone of HRV14 purportedly has been utilized to make single-site mutations in the coat proteins primarily for the purpose of examining the properties of the cell receptor attachment site on the viral surface (Colonno, et al. 1988). However, successful construction of immunogenic chimeras of HRV14, or any other HRV, have not been reported.
An improved way to stimulate an immune response using a chimeric virus would be to utilize a native virus which, (1) is a relatively mild pathogen, such that little risk would be associated with the use of a live chimeric virus vaccine; (2) has a broad range of serotypic diversity (&gt;100 serotypes), lessening the likelihood of preexisting immunity and thereby enabling vaccination in adults; and (3) has the ability to stimulate a significant neutralizing immune response in mucosal membranes as well as in serum. The present invention provides a means for accomplishing this result.